Reconstitution system based on cytosol-depleted cells to study the regulation of phospholipase D

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membrane-associated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes--PLD1 and PLD2--have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5'-[gamma-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.